The present invention relates to the filed of research of substances capable of modifying a function corresponding to a protein or a collection of proteins implicated in a biological process. This type of work, also designated screening, today constitutes an empirical mode of research but particularly for forming new substances, used in numerous laboratories. Among the numerous fields of application of these screening strategies, there can be cited the following examples:
Pharmaceutical laboratories set up libraries of molecules, and research those capable of inhibiting or of slowing down the activity of enzymes implicated in the development of genetic or infectious bacterial or viral diseases.
Following the intensive use of antibiotics, the speed of the appearance of new resistances is currently more rapid than that of the discovery of new antibiotics. Certain hospital laboratories therefore are looking for, always starting from banks of very specific molecules, new antibiotics or new beta-lactamase inhibitors.
Work is equally being carried out to find inhibitors of microorganism multiplication implicated in the biological corrosion of pipes or of containers used in industrial processes.
The present invention has for its object a process for the determination of the activity in vitro of one or several substances using a functional test consisting of detecting and/or measuring a variation of at least one known function corresponding either to one or several proteins produced in vitro in the presence or absence of said substance, or of the substance in the presence or in the absence of proteins produced in vitro. The process of the invention will therefore be designated hereinafter, screening process.
Said known function preferably corresponds to proteins expressed in vitro also called target proteins. The process of the invention therefore aims to determine the activity of the substance tested based on a known function corresponding to one or several protein(s) produced in vitro.
However, in another embodiment of the process of the invention, the known function corresponds to the substance(s) tested. In this case, the process of the invention permits determining if the known activity of this substance is modified by one or several protein product(s) in vitro.
By function is understood the activity of one or several specific proteins still called target proteins of at least one organisms or of at least a process, and that it is detected and/or quantified according to the present invention by the by means of a functional test. Preferably, the target protein is an enzyme. This enzyme can correspond among other things to reverse transcriptase or to the aspartic acid proteinase of the Aids virus, to the Mycobacterium tuberculosis RecA intein, to an antibiotic resistance protein, or also to a collection of enzymes implicated in a metabolic pathway.
By known function is understood the function that is analyzed according to the process of the invention and which can be detected and/or measured by a functional test.
In a particular case of the process of the invention, function is understood as the activity of one or more substances that are detected and/or measured by a functional test.
Variation of function is understood as the difference of the activity of the measured function in the presence or absence of substance. In a second embodiment of the process of the invention, the variation in function corresponds to the activity difference of the substance in the presence of or in the absence of protein expressed in vitro.
The functional test can make use of one or multiple reporter molecules. The reporter molecule can correspond to any molecule capable of directly or indirectly revealing the activity of one or multiple target proteins, and can include a nucleic acid molecule, a protein, a peptide, such as an antibody or a mixture of specific antibodies capable of revealing the activity of a target protein, or such as a substrate or a cascade of substrates, of which one is that of a target protein.
The function can correspond to an enzymatic activity or to an affinity. In the scope of the demonstration of a variation of function corresponding to an enzymatic activity, any type of specific functional test can be contemplated by a person skilled in the art to demonstrate this variation. A person skilled in the art for example can make reference to works such as Methods In Enzymology or Annual Review of Biochemistry, in which a large number of enzyme measuring and substrate preparation methods have been described. In the case of the demonstration of a variation of function corresponding to the affinity for example of an antigen for an antibody, of a protein for DNA, of a receptor for a ligand etc . . . . The demonstration of a variation of function can be carried out for example by tests such as labeled ligand fixation by an isotope or by an enzyme or by a fluorophore, by an immunological detection using antibodies labeled by a metal or by an enzyme or by a fluorophore.
By organism is understood any type of organism or microorganism, such as viruses, bacteria, algae, fungi, or any product containing synthetic or natural nucleic acids permitting the expression of one or more proteins advantageously coding for a function.
By process is understood the development of a disease, of an infection or of cells for example cancers or contaminants or bacterial resistance mechanisms. These processes can take place in a host or in an industrial process (agribusiness, paper treatment, textile).
By polynucleotide substances is understood peptides, proteins, ions, molecules or natural or synthetic chemical compositions, hormones, aromatic compounds, antibodies, antibody fragments, genes, cellular receptors, amino acids, glycopeptides, lipids, glycolipids, sugars, polysaccharides, etc . . . , capable of modifying the activity of one or more functions of an organism or of a process. These listed substances will be capable of corresponding to one or more series heads. Included is any agent capable of modifying the function or functions such as antiviral, inhibitors, stimulants, physico-chemical, radiation or thermal treatments.
The process of the invention is therefore applicable more particularly to the screening of substances capable of modifying the activity of one or more target proteins or function, implicated in a biological process thanks to the in vitro expression of said protein or proteins. The process of the invention permits the screening of multiple substances simultaneously, notably when it relates to substances liable to interact in order to express a function or to modify a function. Consequently, substance must be understood as singular as well as plural of the process of the invention described below.
Similarly, the use subsequently of the term xe2x80x9cfunctionxe2x80x9d in the singular will equally cover the term xe2x80x9cfunctionsxe2x80x9d in the plural and vice versa, except when it is explicitly indicated that it relates to an embodiment of the method of the invention for plural functions.
Thus, the process of the invention permits in a preferred aspect of the invention the screening of substances capable of modifying the activity of a single target protein expressed in vitro having for example an enzymatic activity, such as an amylase, a polymerase, a protease, or capable of modifying an observable property of this protein, such as for example a xe2x80x9cDNA-bindingxe2x80x9d activity, an antigenic activity, or also capable of modifying the activity or a property of a variant of this target protein, such as for example a thermostable amylase, a more processive polymerase, a protease resistance to an anti-protease, or a xe2x80x9cDNA bindingxe2x80x9d protein having a stronger affinity for DNA. But the process of the invention also permits the screening of substances capable of modifying the activity of a collection of proteins expressed in vitro. In this case it is the collection of proteins that make up the target of the substances to test. This collection of expressed proteins can correspond for example to the enzymes making up a synthetic pathway of a metabolite or a degradation pathway of a toxic compound, but it can also correspond to proteins making up the sub-units of a complex protein. The substances to test are therefore then evaluated for their capacity to modify the global activity of this collection of proteins, which implies that these substances can modify either the activity of all, of parts, or of a single one of the proteins of the collection, because the modification of the activity of one or of a part of the proteins leads to the modification of the activity of the collection. The process of the invention also permits the determination or screening in a second embodiment of the invention of the protein or proteins expressed in vitro capable of modifying the activity of one or more known substances.
The quality of a screening technique for substances or proteins capable of modifying the activity of a function rests principally on the effectiveness of the activity test. This test must be highly specific, sensitive, rapid and reproducible. The difficulty of these screening processes in vivo of such molecules rests among other things on the toxicity and the cellular localization (membranal barrier) of the target protein. The present invention aims to offer an isolation process with a high flow of substances or proteins capable of modifying the activity of a target protein or multiple proteins or known function permitting a leveling of the above problems.
This goal is achieved thanks to a process for the determination in vitro of the activity of a substance making use of a functional test, characterized in that a known variation of function is detected and/or measured corresponding either to one or several proteins produced in vitro or to the substance in the presence or in the absence of the proteins produced in in vitro
More particularly, the process of the invention comprises the following steps:
a) the preparation of at least one nucleic acid molecule comprising the gene(s) coding for one or more proteins and the control elements necessary for transcription and translation of the gene(s),
b) The transcription of the nucleic acid molecule(s) prepared at step (a),
c) The in vitro translation of the transcript(s) prepared at step (b),
d) The detection and/or measurement of the variation of a known function corresponding to the protein(s) produced at step (c) in the presence and in the absence of said substance, or to the substance in the presence and in the absence of the proteins produced at step (c).
The substance(s) tested in the scope of the process of the invention are introduced before, after and/or during the transcription and/or the translation of steps (b) and/or (c) and/or the detection and/or measurement of the variation of at least one known function of step (d) of the process of the invention.
In order to facilitate the discussion of the invention, at step (a) of the method of the invention under the term xe2x80x9cgenexe2x80x9d is designated any nucleic acid sequence permitting the expression of the protein(s) corresponding to the detected function(s). It can therefore include a DNA or RNA sequence.
The preparation of one or multiple nucleic acid molecules of step (a) of the process of the invention consists of placing the gene(s) coding for the protein(s) that will be produced at step (c) under the control of elements necessary for the transcription and the translation in vitro, that is to say under the control of a 5xe2x80x2 promoter and possibly of a 3xe2x80x2 RNA polymerase terminator in order to be transcribed in vitro.
A particular embodiment of the invention consists of using the promoter and the terminator of the RNA polymerase of the phage T7 or SP6 or Qxcex2 or xcex.
Similarly, in order to carry out the translation in vitro, a ribosome binding site is introduced upstream of said gene(s).
Thus as previously indicated, the process of the invention allows two embodiments according to which the function detected and/or measured at step (d) corresponds either to the protein(s) produced at step (c) or to the substance. This last case being a particular embodiment of the method of the invention.
In a first embodiment of the process of the invention, the functional test implemented at step (d) corresponds to the detection and/or to the measurement of a variation of known function of the protein(s) produced at step (b) in the presence and in the absence of a substance. The process of the invention is therefore notable in that it permits the screening of substances capable of modifying the activity of one or multiple target proteins (for example a target collection of proteins) expressed in vitro.
The gene(s) coding for the protein(s) correspond to the known function that it is desired to detect and/or measure at step (d) of the process of the invention are known or unknown, synthetic or non-synthetic genes, placed under the control of the sequences described above by classical techniques known to a person skilled in the art.
This or these genes can also be present in a sample containing nucleic acids, such as for example a soil, plant, human, animal, water, microbial culture, cellular, viral, biopsy, organism or process sample. But this sample can correspond equally to products of any method of amplification of genomic DNA, synthetic DNA, mRNA, or any nucleic acid products resulting from treatments currently used by a person skilled in the art. It is well understood that it includes a crude biological sample such as blood, tissues, urine or any other body fluid such as cerebrospinal, synovial, pleural, pericardial, or previously treated in order to prepare the nucleic acids that it contains.
One advantageous form of implementation of step (a) of the first embodiment of the present invention consists of preparing the nucleic acid molecule(s) by an amplification reaction of the gene(s) coding for said protein(s), starting from the sample of nucleic acids. It includes an amplification by PCR or by PCR-derived techniques, of the RT-PCR, nested PCR, multiplex PCR type, or techniques different than PCR of the NASPB, rolling circle or other types. Advantageously this preparation makes use of a couple of oligonucleotides or a couple of primers specific to the nucleic acid molecule(s) comprising the gene(s) coding for the protein(s) corresponding to the function analyzed. This preparation by amplification is carried out with the aid of one or multiple primer pairs, each one composed for example of PCR (FIG. 1) and NASBA:
for the sense primer, some sequence being hybridized upstream of one or multiple nucleic acid molecules comprising the gene(s) coding for said protein(s), and an RNA polymerase primer and possibly a ribosome binding site, and
for the antisense primer, some sequence being hybridized downstream of one or multiple nucleic acid molecules comprising the gene(s) coding for said protein(s), and possibly an RNA polymerase terminator.
Step (a) can be carried out by any other appropriate technique. In effect, the preparation of the nucleic molecule of step (a) can be carried out by any other method known to a person skilled in the art such as a restriction cutting permitting recovery of the gene(s) of interest followed by a ligation directed with the control elements necessary for the transcription and the translation in vitro indicated previously.
As indicated previously, in a second particular form of carrying out the process of the invention, the functional test implemented at step (d) corresponds to the detection and/or to the measurement of a variation of known function of the substance. This form of carrying out, also designated functional genomics, is directed at the measurement of the variation of a known function of the substance in the presence of one or multiple proteins expressed following stages (b) to (c) of the process of the invention.
In this embodiment, step (a) consists of (i) preparing, starting from a sample containing nucleic acids, multiple nucleic acid molecules each one comprising a nucleic acid fragment coming from said sample, associated with a vector molecule, (ii) isolating each nucleic acid molecule composed of one nucleic acid fragment and one vector molecule.
The nucleic acid fragments preferably have a size from 1 to several dozens of kb, preferably from 1 to 40 kb and advantageously from 1 to 10 kb when the sample is of prokaryotic origin. These fragments can carry a partial or whole operon.
The nucleic acid fragments preferably have a size of the order of several dozens to several hundreds of kilobases in the case of a eukaryotic organism. In the particular case where cDNAs are treated at step (a), the fragments will preferably have a size of from 1 to 5 kb in the case of a eukaryotic organism.
The vector molecule is composed of one or several polynucleotide sequences comprising at least one transcription promoter for step (b) and possibly one element facilitating the isolation of the nucleic acid fragment. Advantageously this substance can be one or several streptavidine or biotin molecules, of polypyrol grouping, antibodies, a single or double-stranded polynucleotide sequence, a plasmidic DNA vector preferably not containing sequences permitting the in vivo expression of the associated fragment, or any other compound permitting the isolation of the nucleic acid fragment.
Preferably, the vector molecule is composed of two polynucleotide sequences each one comprising at least one transcription promoter, each one of these sequences being associated at one end with one of the nucleic acid fragments. The transcription promoter(s) carried by the vector molecule are preferably of the strong type.
The vector molecule associated with each fragment of step (b) is advantageously a plasmidic vector preferably not permitting the expression of said fragment in vivo.
In the second embodiment of the invention, the biological sample from which the nucleic acid molecules of step (a) are prepared can come from one or several prokaryotic organisms or eukaryotic cells, or also from identical or different viruses, but it can likewise be composed of a sequence or of a bank of synthetic nucleic acids or also composed of organisms and/or unknown nucleic acids. It can also comprise a eukaryotic DNA bank and then the transcription reaction of step (b) is completed by a splicing reaction and by in vitro maturation of the mRNA by using a nuclear extract.
As indicated above, according to a particular embodiment of the process of the invention, the vector molecule associated with the nucleic acid fragments is a plasmidic vector. In this case, each fragment is inserted in a vector at the level of a cloning site or of a restriction cassette. This plasmidic vector is characterized in that it comprises an RNA polymerase promoter at one side of the cloning site and possibly an RNA polymerase terminator at the other side. It is also possible to envision a vector comprising a cloning site surrounded by two identical or different RNA polymerase promoters and possibly flanked on both sides by a corresponding RNA polymerase terminator or terminators. These promoters and possibly terminators preferably have the characteristic of not functioning in the microorganism that can be used for the separation of recombinant vectors at the step.
In the case where the vector does not possess a promoter or promoters and/or possible RNA polymerase terminator(s), or in the case where the promoter(s) and possible RNA polymerase terminator(s) are not adequate for carrying out step (b), this promoter or promoters and possible terminator(s) can be inserted by any appropriate means. An advantageous implementation of this insertion consists of carrying out a PCR with a set of primers carrying the sequences of the promoter(s) and terminator(s). According to a particular way of implementing the process of the invention, the promoter(s) and possible terminator(s) are of the strong type such as for example those of the T7 or SP6 or Qxcex2 RNA polymerase.
In the case where the vector molecule is a plasmidic vector, the isolation of the recombinant can be carried out by transformation of host cells by the collection of recombinant vectors in a way so as to create a bank of clones, then there is carried out an extraction of the vector or of a part of the recombinant vector contained by each clone of the bank by any appropriate means.
The extraction of the recombinant vector or of a part of the recombinant vector flanked by promoter(s) and possible RNA polymerase terminator(s) can be carried out by any method known to a person skilled in the art, such as by mini preparation and possibly digestion or by PCR. An advantageous alternative consists of carrying out this PCR with oligonucleotides protected at the 5xe2x80x2 end from nucleasic attacks, notably from the nucleases contained in the translation medium, by phosphorothioate groups.
As indicated previously, the isolation of the nucleic acid molecules can be carried out by any physical, mechanical or chemical means such as for example a simple extreme dilution of the collection of the fragments associated with the vector molecule. But the isolation can also advantageously be carried out by using the properties of a specific substance included in the vector molecule, such as an antibody molecule, and the isolation of the fragment is carried out by using the antibody-antigen affinity; or a biotin, and the isolation is carried out by using the biotin-streptavidine affinity, etc . . . .
The screening process according to the invention lends itself to several ways of being implemented notably according to the type of activity of the target protein(s). In effect, the target gene(s) prepared at step (a) can encode for one or several target proteins or functions implicated in diverse types of biological processes. This gene or genes can come from several prokaryotic organisms or from eukaryotic cells or also from viruses. As indicated previously, a process can be for example an infectious disease, a bacterial or viral resistance mechanism, a metabolic chain or also a process implicated in an agribusiness industrial process, of paper treatment, of detergent preparation, of textile manufacturing etc . . . .
Transcription step (b) and the translation phase of step (c) can be simultaneous, which means that the translation phase of step (c) is carried out simultaneously with the transcription of step (b) or decomposed in two distinct steps (b) of transcription and (c) of translation.
The breaking apart of steps (b) and (c) permits optimization of the yields of each step, and thus production of more significant quantities of proteins, which finds its main utility in the case of detection of enzymes of weak specific activity.
This breaking down also permits normalization of the formation of the products at step (c) and of being able to later compare the different functions expressed.
The breaking down between the transcription of step (b) and the translation of step (c) equally permits avoidance of the problems of degradation of the DNA matrix by the nucleases if they were prepared by PCR. In effect, the constituents of the transcription reaction are less contaminated by nucleases, contrary to the translation extracts.
The breaking down moreover permits the use of different translation extracts according to the origin of the targeted DNA. In effect, the translation phase of the transcript at step (c) is advantageously carried out with a translation extract of the same origin or of an origin close to that of the biological sample of which the process of the invention is practiced. Thus, the adequacy between the origin of the translation signals of the transcripts and the translation extract is optimized for optimal translation efficiency. There can be cited by way of example the use of a translation extract of an extremophilic organism if the preparation at step (a) makes use of a nucleic acid sample coming from the same organism or from another extremophilic organism (thermophiles, halophiles, acidophiles, etc . . . ) or also a translation extract of eukaryotic cells if the preparation at step (c) makes use of a eukaryotic nucleic acid sample. These respective extracts are capable of improving the effectiveness of the process. These extracts are chosen for their capacity to translate the transcripts of step (c).
The process of the invention is notable in that it makes use of an adequacy between the expression punctuation of the transcripts of step (b) and the translation extracts used. These extracts are also characterized in that either they do not contain the sought-after function, or they contain it but it is not detectable under the conditions of the test carried out in order to detect the sought-after function. It includes for the example the use of a translation extract containing a mesophilic beta-galactosidase activity permitting translation of a thermophilic beta-galactosidase mRNA and the detection of the activity of the latter at high temperature, which eliminates the mesophilic beta-galactosidase activity.
According to the genetic origin of the genes obtained at step (a), for example DNA of Gram positive, or negative microorganisms, of eukaryotes of viruses, etc . . . , and to the function tested, different translation extracts can therefore be used.
A particular implementation of the process of the invention consists of using at step (c) a translation extract that in fact is a mixture of several translation extracts. It includes for example a translation extract of E. coli overexpressing a chaperon A protein mixed with a translation extract of E. coli overexpressing a chaperon B protein. Any type of mixture can be contemplated once it corresponds to the characteristic described above. In the same manner, it is possible to use a translation extract in which one or several specific tRNAs of one or of several codons is added. The translation extracts thus obtained then permit translation of the mRNA containing these specific codons, such as for example the translation of an mRNA containing an amber codon by adding in the translation extract a tRNA suppressor or suppressors.
The treatment of step (c) with a translation extract can also be carried out with a universal translation extract whatever be the origin of the sample such as for example an E. coli extract and/or any other cellular extract(s) supplemented or not by molecules of interest such as those, for example, previously indicated (tRNA, chaperon . . . ).
It is equally possible to add to the translation extract of step (c) one or several substances favoring a more efficient refolding or maturation of the expressed proteins, such as for example chaperons, detergents, sulfobetaines, membranal extracts, etc . . . .
The detection and/or the measurement of the variation of at least one known function corresponding to the protein(s) produced at step (c) or to the substance is carried out by any functional test known to a person skilled in the art such as defined in the introduction. Step (d) can thus consist of detecting and/or measuring several variations of functions corresponding to one or several of the proteins produced at step (d) or corresponding to one or several substances.
The variations of function(s) are detected our measured in a direct or indirect manner by one or several functional test(s) of the protein(s) produced at step (c) or of the substance(s) to screen. The function(s) is/are read for example continuously for example by fluorimetry or by colorimetry or by viscosimetry or by mass spectrometry . . . .
The detection and/or measurement of the variation of function corresponding to the protein(s) produced at step (c) or to the substance(s) is advantageously carried out at step (d) by a functional test making use of the presence at one of steps (a), (b), (c) or (d) of one or several reporter molecule(s) permitting detection and/or measurement of the activity of the protein(s) produced at step (c) or of the substance(s) to screen corresponding to the function analyzed at step (d).
The process of the invention presents the following advantages as much in its first as in its second embodiment:
The fact of directly working on the activity of the protein(s) produced at step (c) or on the activity of the substance permits good specificity of the screening test.
The sensitivity of the test is explained by the multiplier coefficient of the enzymatic steps (b) and (c), corresponding respectively to transcription and translation, and possibly to the functional test making use of an enzyme.
The process of the invention is fast because entirely automated. For example, a plaque of 384 wells can be treated in 2 to 3 hours.
Finally, the reproducibility is facilitated by the absence of storage of the protein(s) produced at step (c). Additionally, the protein(s) is/are expressed in vitro independently of a complex and not always controllable cellular context (membranal diffusion, cellular localization, cellular toxicity, cellular physiology, induction or repression problems, etc . . . ).
The process of the invention is therefore notable because besides the carrying out of a screening of one or several functions, of a substance it also permits:
development of new functional tests
doing functional genomics.
The process of the invention in effect permits development of new functional tests. We have witnessed these last years a rapid expansion of high flow screening test or xe2x80x9chigh throughput screening (HTS)xe2x80x9d. These screenings, in order to be optimal, require the implementation of sensitive functional tests, but one does not always have these functional tests at one""s disposal. The process of the invention permits the identification of one or several functional test(s) permitting the improvement of the revelation of one or several function(s) of a process or of an organism, and thus the improvement of the measurement of the variation of this function or these functions in the presence or in the absence of one or several substance(s).
Thus, it is possible to develop new functional tests notably of the following fashion:
a) The preparation of at least one nucleic acid molecule comprising the gene(s) coding for one or several proteins and the control elements necessary for transcription and translation of the said gene(s).
b) The transcription of the nucleic acid molecule(s) prepared at step (a).
c) The translation in vitro of the transcript(s) prepared at step (b).
d) The detection and/or the measurement of the variation of a known function corresponding to the proteins produced at step (c) in the presence and in the absence of one or several reporter molecule(s).
e) Thus in the scope of this application the substance corresponds to the reporter molecule capable of revealing a function.
The process of the invention in its first and second embodiment presents a real advantage for the functional genomics.
The process of the invention is more particularly applicable to after-sequencing genomics. The project of decoding the human genome has given birth to a new genomic science, now present in the heart of therapeutic enterprise. Genomics permits identification and description of the genes which direct the manufacture and the putting together of all of an organism""s molecules. These genes coding for the functions of organisms or of processes can be expressed by the process of the invention, which permits confirmation of the function encoded by a reading frame located by bioinformatics, of highlighting one or several pharmaceutical substances capable of modifying said function encoded by one or several proteins expressed by the process of the invention or which will permit identification of the targets of a substance corresponding to one or several functions of an organism or of a process expressed at step (c).
In 100 years of existence, the pharmaceutical industry has identified several hundreds of receptor sites. With functional genomics, other existing receptor sites can be attained, as many potential target functions for future substances that remain to be contemplated.
By way of example of such an application, the spotting by bioinformatics of an ORF possibly encoding a target, confirmation of this target by the process of the invention and finding for it substances which can make its activity vary can be cited.
Another example of functional genomics consists of identifying at the genetic level a gene corresponding to a protein whose activity is modified by one or several substances. In the case where the effect of a pharmaceutical substance on an organism or a process is known, this application of the process of the invention presents a particular interest. The genes corresponding to the proteins encoded by this organism or this process will then be prepared according to a particular implementation of step (a) described previously, in a manner so as to isolate said genes and express them at steps (b) and (c) of the process of the invention. A functional test at step (d) permits measurement of the activity of the substance in the presence and in the absence of each one of the proteins expressed at step (c). The measurement at step (d) of a variation of activity of the substance in the presence and in the absence of proteins expressed at step (c) then permits identification of the protein(s) whose activity is modified by said substance within the organism or the process.
This embodiment of the invention presents the enormous advantage of being able to go directly up to the gene starting from the protein(s) expressed at step (c) and isolated at step (a). The functional target can thus be identified at the genetic level of one or several substances.
Each time that a functional test can be implemented (enzymatic test, binding test, etc . . . ), the process of the invention permits high flow subjecting of the collection of the expressible proteins of a considered organism to an automated functional test and of going up in a few hours to the gene corresponding to the sought-after function.
The description which follows preferably refers to examples of carrying out the first embodiment of the process of the invention in which the protein(s) corresponding to the function(s) detected and/or measured at step (d) are designated target protein(s) and the corresponding coding gene(s) is/are designated target gene(s). Thus, the process of the invention permitting screening of the substances capable of modifying the activity of a target protein or of several target proteins expressed in vitro includes the following steps:
a) the preparation of at least one nucleic acid molecule comprising the gene(s) coding for the target protein(s) and the control elements necessary for the transcription and the translation of said target gene(s).
b) the transcription in vitro of the nucleic acid molecule(s) prepared at step (a).
c) the translation in vitro of the transcripts of step (b).
d) the detection and/or the measurement of the variation of at least one known function corresponding to the target protein(s) produced at step (c) in the presence and in the absence of the substance to screen.
As previously indicated, the process of the invention permits detection and/or measurement at step (d) of the variation of one or several functions corresponding to one or several target proteins produced at step (c). It refers for example to proteins expressed by an operon. Consequently, the process of the inventions lends itself to several forms of being carried out.
When it relates to the screening of substances capable of modifying a function corresponding to the activity of a target protein, the process of the invention includes the following steps:
a) the preparation of a nucleic acid molecule comprising the gene coding for said protein, 5xe2x80x2 of said gene an RNA polymerase promoter and a ribosome binding site and possibly an RNA polymerase terminator 3xe2x80x2 of said gene.
b) the transcription in vitro of the nucleic acid molecule prepared at step (a),
c) the translation in vitro of the transcripts of step (b),
d) the detection and/or the measurement of the variation of at least one known function corresponding to the target protein(s) produced at step (c) in the presence and in the absence of the substance to screen.
But the process of the invention can also be applied to the screening of substances capable of modifying the function corresponding to a collection of target proteins. The genes coding for these proteins can be located on the same DNA fragment as in the case of an operon, or at different places of the genomic DNA as in the case of certain metabolic pathways.
When the process of the invention relates to the screening of a substance capable of modifying the activity of a collection of proteins, step (a) of the process lends itself to the following two embodiments:
i) Either, the genes are grouped together under the form of an operon, and then step (a) consists of preparing a nucleic acid molecule comprising the genes (the operon) coding for the proteins, 5xe2x80x2 of the collection of said genes (of the operon) an RNA polymerase promoter, possibly 3xe2x80x2 of the collection of said genes (of the operon) an RNA polymerase terminator, and for each of said genes its natural ribosome binding site.
ii) Or, said genes are separated and then step (a) consists of preparing one or several nucleic acid molecules comprising the genes coding for the proteins, 5xe2x80x2 of each one of said genes an RNA polymerase promoter and a ribosome binding site, and possibly 3xe2x80x2 of each one of said genes an RNA polymerase terminator.
In the first embodiment (i) above, the ribosome binding site of each one of the genes is its natural ribosome binding site, and it is then preferred to use at step (c) a translation extract prepared starting from the organism from which the target gene(s) come from or from a phylogenetically close organism.
In the second embodiment (ii) above, the ribosome binding site can be the natural site of each one of the genes or another ribosome binding site more adapted to the translation step (c).
A variant of the first (i) and of the second (ii) embodiment of the above process consists of carrying out in parallel or simultaneously the previously described process of the invention with a single protein, each step (a) being carried out with each one of the genes. An alternative to the parallel or simultaneous carrying out of the process of the invention, consists of separately carrying out for each one of the genes the steps (a), (b) and (c), then, for the final screening implicating each one of the proteins, gathering the products of the steps (c) together in order to carry out step (d).
Similarly, when the process relates to screening substances capable of modifying the activity of a collection of proteins in which the genes are physically separated on the organism""s genome, the process of the invention consists of carrying out in parallel or simultaneously, the process of the invention previously described with a single protein, each step (a) being carried out with each one of the genes. An alternative to the carrying out in parallel or simultaneously of the process of the invention, consists of separately carrying out for each one of the genes, the steps (a), (b) and (c) then, for the final screening implicating each one of the proteins, to collect the products of the steps (c) in order to carry out step (d).
As previously indicated, the process of the invention permits screening of the substances capable of modifying the activity of a collection of target proteins. The process of the invention can therefore be applied to the screening of substances capable of modifying the activity of the variants of a protein or of the variants of a collection of proteins. The gene(s) corresponding to this variant or these variants can be contained for example in the same nucleic acid sample from the start. By way of example of this application of the process of the invention, the different mutants of the VIH protease gene contained in a sample of a patient infected by this virus can be cited. The implementation of the process of the invention then consists of carrying out each step (a) with each one of the mutants of said gene in such a way as to express each one of them separately. The separation of the mutants contained in the sample can be carried out by cloning, extreme dilution, or by any other method known to a person skilled in the art. This application of the process of the invention therefore consists of screening all of the substances to test not on a target protein or on a collection of target proteins but on all the existing variants of them, of which the corresponding genes are contained in a single sample of nucleic acids. This application of the process of the invention therefore permits the determination of the spectrum of action of the substances to test.
The invention therefore also relates to the application of the screening process to substances capable of modifying the different variants of a target protein or of a collection of target proteins. In effect, as indicated above for the VIH protease, the sample of a patient infected by this virus can contain several variants of the virus each one expressing a different protease. It is therefore interesting to carry out a screening on each one of the different proteases in order to determine those that are inhibited by the known antiproteases or those that can be by new substances to test.
On the basis of this example of the HIV virus, the invention permits the analysis in vitro of the different variants of a target protein or of a collection of target proteins. This goal is attained according to the invention, by possibly amplifying the different mutants, for example by cell culture or by molecular amplification, then by isolating each mutant gene, for example by cloning or by extreme dilution, and by expressing each one of these genes in accordance with steps (a), (b) and (c) of the process of the invention, and finally by screening the substances capable of modifying the activity of the protein(s) produced during step (d). The screening process consists for example in the case of the VIH proteases of carrying out an inhibition test with the different inhibitors or the different substances to test on each protease individually. The activity of each one of the proteases expressed in accordance with the process of the invention can then be characterized in such a fashion as to reveal the representation of the different variants of the virus that infect a patient. The result of the screening process according to the invention then permits adapting the therapy of the patient.
As stated previously, the functional test can be used for one or several reporter molecules. In a first particular embodiment of the process of the invention, the reporter molecule must be present at step (c) or (d) to reveal the possible activity of the target protein according to the role of the substance tested during the screening.
But the reporter molecule can also be present in the reaction mixture from one of the steps (a) to (b), either under its final reporter molecule form, or, according to a particular embodiment of the invention, under the form of a nucleic acid molecule (DNA or RNA) corresponding to the gene coding for said reporter molecule, and then designated hereinafter reporter gene. In this particular embodiment, the reporter molecule will advantageously be produced during step (c) conjointly with the target protein(s).
Thus, according to a particular embodiment of the screening process of the invention, the reporter molecule is a protein that is produced during step (c) conjointly with the target protein(s). Advantageously, in this embodiment of the process of the invention, the gene coding for the reporter molecule is placed under the control the transcription and translation regulation sequences similar to that of the gene(s) coding for the target protein(s), in a fashion such that the reporter gene is co-expressed with the target gene(s).
By way of example, the reporter gene can be the gene of the GFP protein (Green Fluorescent Protein) or that of the beta-lactamase (TEM-1). In the case of the GFP, it is the fluorescence emission that is evaluated. It is then possible to carry out a test such that the GFP only fluoresces if the activity of the products of the target gene(s) is modified by the molecule(s) tested. In the case of the beta-lactamase, it is the activity of this enzyme that is evaluated by incubating a fraction of the translation reaction in a buffer containing nitrocephine. Nitrocephine is a chromogenic beta-lactamine that has the property of changing color from yellow to red when it is hydrolyzed by an active beta-lactamase. A yellow tests indicates for example that the molecule(s) tested is(are) capable of modifying the activity of the target gene product.
Any other reporter gene can be contemplated in the process of the invention, such as those of beta-galactosidase, luciferase, peroxidase, or a microperoxidase, etc . . . . It is worth noting that the GFP reporter gene has the advantage of producing a protein whose activity is instantly measurable, which permits a supplemental savings in time. But, a reporter gene coding for a protein having a beta-lactamase type enzymatic activity has a large sensitivity due to the enzymatic multiplier coefficient.
A particular embodiment of the process of the invention with a reporter gene consists of expressing in vitro a reporter gene within which one or several genes coding for the protein(s) have been inserted, the collection of these genes being under the control of the same transcription and translation regulatory sequences.
An example of a nucleic acid molecule prepared at step (a) corresponding to this embodiment of the process of the invention is represented at FIG. 2 attached. This particular case, where the target gene and the reporter gene are co-expressed, is quite adapted to the target gene corresponding to the inteins such as the Mycobacterium tuberculosis RecA intein.
Another particular embodiment of the process of the invention with a reporter gene consists of using a target gene (coding for a target protein or a function) that can itself be a reporter gene. An example of a nucleic acid molecule prepared at step (a) corresponding to this embodiment of the process of the invention is represented at FIG. 3 attached. This embodiment is particularly adapted to a target protein having a directly detectable activity in vitro, such as an enzymatic activity, such as beta-lactamase. The beta-lactamase gene plays an essential role in the resistance to beta-lactamine type antibiotics by hydrolyzing these molecules before they have been able to act on their therapeutic targets. Thus, once the enzyme has been put in the presence of the molecules to test at step (b), (c) or (d), the beta-lactamase activity can be evaluated thanks to a nitrocephine test.
The steps of the process of the invention can be carried out successively without interruption by the same operator, advantageously on an automated device integrating each one of the steps, or can be carried out discontinuously, possibly by different operators.
If the tested substance corresponds to a polynucleotide sequence, it will be able to have a role as such or as a gene coding for a protein. In this latter case, this polynucleotide sequence possesses all the sequences necessary for its in vitro expression during the transcription and translation reaction. Preferably, it will be expressed by the same regulatory sequences as the target gene(s) in order that their expression will be concomitant and that the molecule will have the time to modify the activity of the products of the gene(s) expressed after steps (b) and (c). This avoids generation of false positive or false negative results.
One of the possible roles of the substances to screen is to slow down, even to stop, in vitro, the activity of a target protein or of a collection of target proteins, which can be implicated for example in the progression of a disease. These molecules will for example be resulting from libraries having a particular pharmacological interest, resulting from work in combinatorial chemistry taking into account the structure of pre-existing productive inhibitors and/or the structure of the target enzyme active site and its particular characteristics.
But the tested substances in the scope of the process of the invention can equally have a stimulator or activator effect on the activity of the product of one or several target genes having a key role in processes. Such molecules can prove useful insofar as enzymatic cofactors permitting reduction of the enzyme activity in a given industrial phenomenon (agribusiness or other) and thus to increase in yield.
Each time that a tested substance is proven effective for modifying the activity of the target protein, it is advantageous to verify, by an appropriate method, that this substance does not inhibit one of the steps of the process. Moreover, after having identified a substance capable of modifying the activity of a target protein, this protein can be tested according to the process of the invention on proteins having an activity similar to that of the target protein(s).
The process of the invention can advantageously be implemented on any type of support and is therefore easily capable of being automated. By support is understood for example microtitration plaque wells and equally biochips. They can contain several dozens to several thousands of sites. Thus, on a single support from dozens to thousands of substances will be able to be tested for their capacity to modify the activity coded by the target gene(s) of one or several nucleic acid molecules prepared at step (a).
A particular embodiment of the process of the invention consists of charging the wells of these plaques with a coupled transcription and translation reaction mixture (components necessary for transcription and translation) containing one or several nucleic acid molecules prepared at step (a) and the reporter molecule, then to freeze them to avoid the premature initiation of the transcription/translation reaction. The frozen plaques are placed on an automatic machine, which deposits in each well a volume of the molecule to test permitting dilution of the transcription and translation mixture until it is concentrated 1 time. A homogenization can be contemplated through the automatic machine, but the reaction volumes should be sufficiently small in order for the diffusion to assure this homogenization.
The measurement of the activity of one or several target proteins or of the reporter molecule of step (d), and therefore of the effect of the tested substance on the target protein or on a collection of target proteins, can be equally advantageously automated. The possible modification of the target protein activity is directly read on the support in a fluorimetry reader (if the reporter is for example GFP) or in a fluorimetry reader (if the reporter is for example beta-lactamase). The automated readings will be adapted for the revelation of the reporter. It is thus possible to carry out a reading of the reporter activity continuously.
The automation presents advantages in terms of the potential screening. The process of the invention can be contemplated in a micro-manufacturing concept in such a manner as to manipulate on a unique support thousands of reaction nanovolumes. This represents a certain advantage in terms of cost but equally in high flow screening potential. The process of the invention implemented on a biochip permits screening of collections of substances for example resulting from combinatorial chemistry work having for example antifungal or antibacterial or antiviral or anticancer properties or from collections of substances directed against diseases such as Parkinson""s or Alzheimer""s. The process of the invention applied at high flow permits identification of the substance key best adapted to a function lock given in a minimum time. It therefore permits the rapid and systematic testing of all of the possible combination of a set of given substances.
Consequently, the invention relates to a device comprising an arrangement of one or several supports, of robots and of a reader of said supports for the carrying out of the stages of the process of the invention.
Finally, the invention has for an object a kit for screening substances capable of modifying the activity of one or several functions in vitro in accordance with one of the embodiments of the process of the invention.
In a first embodiment, such a kit includes the means for revealing the function, an RNA polymerase, nucleotide sequences for the preparation of nucleic acid molecules comprising the gene(s) permitting the expression of the protein(s) corresponding to the detected and/or quantified function, the four triphosphate nucleotides, the mixtures necessary for said preparation, for the transcription and the translation, possibly some substances, possibly some controls.
In a second embodiment, a kit according to the invention comprises:
possibly substances necessary for the preparation of the nucleic acid molecules comprising the gene(s) permitting the expression of the protein(s) corresponding to the quantified and/or detected function,
any support such as a microtitration plaque or chip containing: the means for revealing the function, an RNA polymerase, the four triphosphate nucleotides, the transcription and translation mixtures, possibly some controls.
The kits and the supports can be contemplated for the detection and/or the measurement simultaneously or not of a variation of one or several function of one or several processes or of one or several organisms.
The invention therefore also has as an object a support having a series of sites for the implementation of a method of the invention, characterized in that each one of said sites permits the detection and/or the measurement of a variation of function.
The substance to screen can be present in the wells or can be added at the end of the reaction.